Pigment composition, spectral characterization and photosynthetic parameters in Chrysochrom ulina polylepis

Transcription

1 Vol. 83: MARINE ECOLOGY PROGRESS SERIES Mar. Ecol. Prog. Ser. Published July 16 Pigment composition, spectral characterization and photosynthetic parameters in Chrysochrom ulina polylepis Geir ~ohnsen', Egil Sakshaugl, Maria vernet2 ' Trondhjem Biological Station, The Museum, University of Trondheim, Bynesveien 46, N-7018 Trondheim, Norway Marine Research Division, A-018. Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92093, USA ABSTRACT: The photobiological response of an isolate of the prymnesiophyte Chrysochromulina polylepis, obtained from a bloom in the Skagerrak in May-June 1988, was evaluated with respect to pigment composition, spectral dependence of light harvesting, and photosynthetic parameters of cultures grown at 75 to 120 gm01 m-2 S-' irradiance, 16 h day length and 15 C. Results were compared to similarly grown cultures of the diatom Skeletonerna costatum that appeared before and after the C. polylepis bloom. Chl a-specific absorption of light ("a,) and chl a-specific absorption of quanta transported to photosystem 11, estimated by means of a scaled fluorescence excitation spectrum ("F), were 1.7 to 2.1 times larger in C. polylepis than in S. costatum in the visible spectrum. C. polylepis harvested blue-green light (450 to 500 nm) particularly efficiently. This is related to a high proportion of 19'- hexanoyloxyfucoxanthin and chl c3 relative to chl a. Nonetheless, both C. polylepis and S. costatum absorb light more efficiently in 'clearest' blue ocean water than in 'clearest' green coastal water according to calculations based on spectrally corrected absorbed quanta transported to photosystem I1 ("F). Carbon-specific light absorption was about the same in the 2 species since the chl a: C ratio in S. costatum was twice as high as in C. polylepis. C. polylepis had a much smaller maximum carbon uptake (P:) than S. costatum. Differences between the 2 species in terms of photosynthetic parameters, pigment composition, and spectral characteristics normalized to chl a, carbon, and cell are discussed. INTRODUCTION The bloom of Chrysochromulina polylepis in the Skagerrak in 1988 was first observed after a diatom bloom dominated by Skeletonema costatum along the Swedish coast in the first week of May (GranBli et al. 1989). For the next 2 wk C. polylepis bloomed in the Skagerrak and was transported along the sniltharn coast of Norway. The population of C. polylepis was observed at the pycnocline, often situated at 5 to 10 m depth. The maximum cell concentrations were 40 to 80 X 106 cells 1-' (Aksnes et al. 1989, Lindahl & Dahl 1989). During the bloom period the weather was exceptionally bright and sunny, the waters were extremely stratified, and the temperature in the surface layer was 6 to 12 C (Aksnes et al. 1989, Skjoldal & Dundas 1991). The bloom harmed marine life and aquaculture (Dahl et al. 1989). When the C. polylepis bloom terminated near Arendal (Norway), a bloom of S. costatum developed near the surface (Skjoldal & Dundas 1991). It has been suggested that prymnesiophyte blooms in the KattegatKkagerrak and along the southern coast of Norway occur when limitation by silicate prevents diatoms from forming large stocks (Aksnes et al. 1989, Kaartvedt et al. 1990). The light regime, however, may also play a role, particularly in the initial phase of a bloom before nutrients become limiting. During May, irradiance levels were above the mean values for that month measured in earlier years, and the largest positive anomaly occurred from 6 to 12 May, which coincided with the first registration of Chrysochromulina polylepis (Skjoldal & Dundas 1991). Waters in the Skagerrak and along the Norwegian coast differ from oceanic waters in that their colour is blue-green to green, mainly due to high concentrations of humic substances (Jerlov 1976). Spectral irradiance may have an impact on species distribution. To evaluate the photobiological response of Chrysochromulina polylepjs, with respect to light, including its spectral distribution, we investigated pigment compos- C9 Inter-Research 1992

2 242 Mar. Ecol. Prog. Ser. 83: , 1992 ition, spectral dependence of light harvesting, and photosynthetic parameters of moderately shadeadapted cultures. These results were then compared to results for Skeletonema costaturn, grown in the same light regime and temperature. MATERIAL AND METHODS Culture conditions. The Chrysochrornulina polylepis (Manton & Parke 1962) strain was isolated from a bloom that occurred in May-June 1988 outside Hvaler (outer Oslofjord, Norway: 59'001N, 10 45'E). C. polylepis was maintained in f/2 medium (Guillard & Ryther 1962) at 15"C, 34 ppt salinity and 16 h day length. Scalar irradiance (E,, PAR) was 75 or 120 pm01 mp2 S-' (provided by 4 Philips TL 40W/55 fluorescent tubes). E, was measured with a QSL-100 quantum sensor (Biospherical Insirumenis). Pigment and cilemical coiilposition as well as light absorption characteristics did not differ at the 2 irradiances, and we here report the pooled results. Skeletonema costaturn, clone Skel-5, isolated from the Trondheimsfjord (Norway) in 1969, was grown at 75 pm01 m-2 S-'. Pigments. For pigment extraction, cultures were concentrated on Whatman GF/C glass fibre filters at 50 mb differential pressure. The filters were then extracted for 20 h at 4 "C in darkness in 90 O/O acetone bubbled with N2 (Chrysochromulina polylepis) or according to Hallegraeff (1981) for Skeletonema costatum. Extracts were cleared by filtration through Whatman GF/C filters and injected into the HPLC column without further treatment. The chlorophyll a (chl a) concentration was also estimated spectrophotometrically according to Jeffrey & Humphrey (1975) with the same pretreatment as above. Spectrophotometrical estimation of chl a was used for normalizing the light absorption spectra and chl a: C ratios and were in agreement with the HPLC values. Pigments were analyzed by high-performance liquid chromatography (Merck & Hitachi L-6200 HPLC) on a SPHERI-5 RP-18 reverse-phase C-18 column (Brownlee Labs 25 cm X 4.6 mm, 5 pm particles) by elution in a low-pressure gradient system consisting of a linear gradient from 100 O h A to 100 O/O B in 10 min and maintaining B for another 15 min. Solvent A (11) consisted of 80 : 20 methanol : water (v: v) where 100 m1 of distilled water was prepared with 1.5 g tetrabutylammonium acetate and 0.96 g ammonium acetate as ionpairing agent (Mantoura & Llewellyn 1983). Solvent B consisted of 60 : 40 methanol : ethyl acetate (v: v). Chlorophylls and carotenoids were monitored by absorption at 440 nm and quantified by calibration of the column with pigments isolated by thin-layer chromatography from a culture of the diatom Thalassiosira nordenskioeldii. For chlorophylls we used the extinction coefficients of Jeffrey & Humphrey (1975) and Jeffrey & Wright (1987). For carotenoids, extinction coefficients were as follows - fucoxanthin and its derivatives: g-' cm-' at 450 nm; diadinoxanthin, diatoxanthin and their derivatives: g-1 cm-' at maximum absorption (446 to 453 nm) (Jeffrey 1968, Davies 1976). Absorption spectra of the eluted pigments were recorded in the eluent on a mtach Spectrophotometer Model U-2000 fitted with a flowthrough cell and compared to published spectra for identification (Davies 1976, Wright & Shearer 1984). A more detailed analysis of the carotenoids of Chrysochrornulina polylepis has been published elsewhere Table 1. Chrysochromulina polylepis. Spectral characteristics and cellular contents of pigments. Parentheses denote shoulders in the absorption spectra Pigment Reference Absorption Cellular % Total time maxima concentration carotenoid (min) (eluent) (pg cell-') (W: W) chlorophyll^^ ,587, Chlorophyll c$ ,586, Fucoxanthin , (468) '-hexanoyloxyfucoxanthin , gb, 473 9' cis hexanoyloxyfucoxanthin , Diadinoxanthin 11.4 (423), 446, Diatoxanthin , '-hexanoyloxyparacentrone 3-acetate 12.1 (422), 446, Chl a ,618, (3-Carotene 20.2 (4281, 454, (422)', 447, 472 May include chl c,, absorption maxima in ethanol, C in ether

3 Johnsen et al. Photobiology of Chrysochromulina polylepis 24 3 TIME (SECONDS) Figure 1. Chromatogram (absorbance at 440 nm) of pigment extract obtained from (A) Chrysochromulina polylepis and (B) Skeletonema costaturn. Peak identifications: 1 = chl c3, 2 = chl c,+z, 3 = fucoxanthin, 4 = 19'-hexanoyloxyfucoxanthin, 5-9'cis hexanoyloxyfucoxanthin, 6 = diadinoxanthin, 7 = diatoxanthin, 8 = 19'hexanoyloxyparacentrone 3-acetate, 9 = chl a. l0 = p-carotene (Bjerkeng et al. 1990). Our system separates chl c3 from chl cz well (1 min between peaks; Table l), but chl c2 and cl would coelute if the latter were present (Fig. l). In vivo spectral characteristics. Chl a-specific light absorption ("a,) was measured on Whatman GF/C filters according to Mitchell & Kiefer (1988). Chl a- specific fluorescence excitation spectra were measured at an emission wavelength of 730 nm (Neori et al. 1988) and were quantum-corrected by means of the dye Basic Blue 3 (Kopf & Heinze 1984). They were then scaled by matching of the red peak of the fluorescence excitation spectrum at 676 nm to the corresponding absorption peak of "a, to provide estimates of specific absorption of quanta transported to photosystem 11, where O2 is released ["F, m2 (mg chl a)-'; Sakshaug et al "F may be interpreted as an action spectrum fcr photosynthesis (Haxo 1985, r\!eori et a!. 1988). "3, and "F were measured in duplicate at 1 nm intervals. The integrated values of "Z, and "F (400 to 700 nm) - and thus the photosynthetic efficiency (2 =, $, "F, where,, G, = maximum quantum yield) depend on the spectral distribution of ambient light and were calculated according to More1 et al. (1987): where "Z, = absorbed quanta, spectrally corrected; "F = absorbed quanta transported to photosystem 11, WAVELENGTH (nm) Fig. 2. (A) Relative spectral irradiance [xlo3. E, (PAR) = l] at 5.20 and 50 m depth in 'clearest' green (G) and blue waters (B). (B) Spectrally corrected chl a-normalized absorption of quanta transported to photosystem 11 [OF(A) Eo(l), m' (mg chl a)-' X 103] in 'clearest' green and blue water at 20 m depth assuming infinitesimal chl a concentration. E,(PAR) = 1. (C) As (B), but carbon-normalized ['F(A) Eo(A), m2 (mg C)-' X 106]. Cp: Chrysochrornulina polylepis; Sc: Skeletonema costaturn spectrally corrected; X (A) = "a, (1) or "F (1); E, (A) = in situ spectral irradiance; E,(PAR) = integrated irradiance. Calculations of spectral irradiance vs depth (Fig. 2A) were carried out on the basis of spectral vertical diffuse attenuation coefficients for 'clearest' green water in the Trondheimsfjord on 6 March (< 0.1 mg chl a m-3) and

4 244 Mar. Ecol. Prog. Ser. 83: , 1992 'clearest' blue ocean water (Smith & Baker 1981) using the daylight spectrum (cloudless sky) in Trondheim (63" N) at noon on 6 March. Spectral irradiance in 'clearest' green water was measured with a Li-Cor 1800-UW spectroradiometer (400 to 700 nm) at 1 nm intervals and is very comparable with the colour of Skagerrak water (R. Dall~kken pers. comm.). Cellular carbon and nitrogen were analyzed in a Carlo Erba Elemental Analyzer, model 1104, after treatment with fuming hydrochlorid acid. Photosynthetic parameters. P vs I experiments were performed at 15 C in 'blue-green' Light provided by 4 Philips TLM 115W/33RS fluorescent tubes; for spectral characteristics, see Johnsen & Hegseth (1991). Two prefiltered ampoules with NaHt4C03 activity of 370 kbq ml-' (New England Nuclear, NEC-086s) were added to 80 m1 algal culture. Duplicate 1 m1 samples were incubated 4 h into the light phase for 1 h in 20 m1 scintiiiaiion vials (Zirlsser poiyelhylene) in a modiiied 'Photosynthetron' (Lewis & Smith 1983) with 20 different irradiances (7 to 565 pm01 m-'s-'; Sakshaug et al. 1991). This technique involves measurement on unfiltered samples and therefore does not discriminate between particulate and dissolved phases of 14C. PVS I parameters were computed according to Platt et al. (1980) by means of the curvilinear least-square regression program LSQUARE (see Table 3 for definitions and units). RESULTS Pigments Predominant accessory pigments of Chrysochromulina polylepis are chlorophyll c3 (Jeffrey & Wright 1987) and chl c2 in addition to 19'-hexanoyloxyfucoxanthin as the major carotenoid, while fucoxanthin contributes 13 O/O of total carotenoids (Table 1, Fig. 1). In contrast, fucoxanthin is the major accessory pigment in Skeletonema costatum (Pennington et al. 1988; Fig. 1, Table 2). The pigment composition of C. polylepis is similar to that of the Prymnesiophyte Emiliana huxleyi (Wright & Jeffrey 1987), but differs from the Plymouth isolate of C. polylepis because 2 recently found pigments in our strain (Bjerkeng et al. 1990) are absent in that strain (Riley & Wilson 1967). Similar to the findings of Riley & Wilson (1967), our strain exhibits minor amounts of a carotenoid eluted after diatoxanthin (Table 1. Fig. 1; at 12.1 min) with absorption maxima at 422, 446 and 471 nm. This carotenoid was identified as 19'-hexanoyloxyparacentrone 3-acetate (Bjerkeng et al. 1990). Also, a minor carotenoid which is eluted before diadinoxanthin was found (Table 1, Fig. 1; at 11.0 min). It has absorption maxima at 448 and 468 nm Table 2. Cellular contents of pigments in Skeletonerna costaturn; chl c: chl a and fucoxanthin:chl a ratios in Chrysochromulina polylepis and S. costaturn Pigment Cellular conc. % Total (pg cell-') carotenoid (W:W) Chl c Fucoxanthin Diadinoxanthin Diatoxanthin Chl a 0.83 p-carotene C. polylepis S. costatum Chl c: chl aa Fucoxanthin : chl a 0.58~ 0.52 a Chl c = total chl cfor each species 19'hexanoyloxyf.ricoxanthiii is included and has been identified as 9'cis hexanoyloxyfucoxanthin (Bjerkeng et al. 1990). In vivo spectral characteristics The in vivo chl a-specific absorption spectrum ("a,) of Chrysochromulina polylepis exhibits major peaks at 439, 468, 592, 639 and 675 nm, which correspond to chl a (439, 639 and 675 nm) and chl c2+c3 (468, 592, and in part 639 nm; Fig. 3A). A shoulder at 500 to 540 nm probably corresponds to in vivo absorption by the carotenoids 19'-hexanoyloxyfucoxanthin and fucoxanthin, which may also contribute significantly at 468 nm. The scaled fluorescence excitation spectrum "F of Chrysochromulina polylepis exhibits a maximum at 472 nm, which corresponds to Ilght-energy transport from 19'-hexanoyloxyfucoxanthin, chl c2 and in particular chl CS, which have maximum absorption at 457 nm in solution (Table 1, Fig. 3B). On a chl a-normalized basis, Chrysochromulina polylepis absorbs Light far more efficiently than Skeletonema costatum: "a, (676 nm) is m2 (mg chl a)-' in the former vs in the latter (Fig. 3A). Corresponding values for 440 nm are and m2 (mg chl a)-' (Fig. 3A). Photosynthetic parameters Chrysochromulina polylepis exhibits a maximum photosynthetic rate (P:) of 1,3 mg C (mg chl a)-' h-'. This rate compares well to in sltu assimilation num-

5 Johnsen et al.: Photobiology of Chrysochron~~~lina polylepjs 245 DISCUSSION Pigments WAVELENGTH (nm) Fig. 3. Chrysochromulina polylepis (Cp) and Skeletonema costatum (Sc). (A) Chl a-specific absorption ("a,) and scaled fluorescence excitation spectra ("F). (B) In vitro absorbance of the individual pigments isolated from C. polylepis and scaled to the cellular concentration presented in Table 1: (A) chl a, (B) chl c3, (C) chl C?, (D) 19'-hexanoyloxyfucoxanthin, (E) fucoxanthin, (F) diadinoxanthin (solvent; eluent from the HPLC column) bers (pb) of 0.3 to 1.9 mg C (mg chl a)-' h-' for C. polylepis at the pycnocline in the southern Skagerrak on 30 May to 1 June 1988 (Nielsen et al. 1990). P: for Skeletonema costatum, however, is much higher [4.7 mg C (mg chl a)-' h-'; M. Gilstad & G. Johnsen unpuhl.]. Phntnsynthetic efficiency (2) and the photoadaptation index (Ik) for C. polylepis in incubator illumination was mg C (mg chl a)-' h-' (W01 m-2 S-') and 48 pm01 m-2 S-', respectively. The 2 value in S. costatum was almost the same as that for C. polylepis mg C (mg chl a)-' h-' (pmol m-2 S-')], while Ik was 4.4 times higher in the former (211 pm01,-z S -I). In C. polylepis, the photoinhibition index (pb) and the photoinhibition irradiance (Ib) were 0.9 X IO-~ mg C (mg chl a)-' h-' (pmol m-2 S-'), and 1700 pm01 m-2 S-', respectively (Table 3). Thus, in contrast to S. costatum, no photoinhibition of photosynthesis was registered (Table 3). A comparison with other bloom-forming prymnesiophytes from western Europe demonstrates that Chrysochromulina polylepis 1s similar to Emiliana huxleyi, Phaeocystis pouchetii and Pryrnnesium parvurn in having chl c3 as well as chl c2 (Jeffrey & Wright 1987, Fawley 1989), and to E. huxleyi and Corymbellus aureus (Gieskes& Kraay 1986) in the dominance of 19'- hexanoyloxyfucoxanthin. It differs, however, from E. huxleyi by not having a phytol-containing chl c derivative (Nelson & Wakeham 1989). As expected for shade-adapted Chrysochromulina polylepis, the photosynthetically active pigments (chl a, c2, and c3, 19'-hexanoyloxyfucoxanthin and fucoxanthin) are abundant while carotenoids believed to be involved in photoprotection (diadinoxanthin and diatoxanthin; Haxo 1985, Vernet et al. 1989) are low with 13 % by weight of the total carotenoid in the cell (Table 1). Diadino- and diatoxanthin contributed 8 % of total carotenoids in shade-adapted Skeletonema costatun], and 2.5 to 15 % in shade-adapted Barents Sea diatoms (Sakshaug et al. 1991, G. Johnsen unpubl.; Table 2, Fig. 1). We found a chl a content of 0.93 pg cell-' in Chrysochrornulina polylepis. This indicates that our experiments are representative of cells living at the pycnocline. This chl a content belongs to the high range of data for the Skagerrak bloom in May/June 1988 (0.15 to 1.0 pg cell-'; Dahl et al. 1989, Lindahl & Dahl 1989, Nielsen et al. 1990). The chl a content in Skeletonema costatum is comparable to that of C. polylepis, 1. e pg cellp'. This value is comparable to the chl a content (0.85 pg cell-') according to earlier studies of the same clone of S. costatum grown at 70 ~imol m-' S-', 15"C, and 10 h daylength (Sakshaug & Andresen 1986). In vivo spectral characteristics The fluorescence maximum at 472 nm (Fig. 3A) is not evide~t ir! "a, afid may represen! a particu!ar!y efficiezt transfer of light-energy from 19'-hexanoyloxyfucoxanthin, chl c2 and chl c3 to reaction-center chl a in photosystem 11. Other fluorescence excitation peaks coincide with the absorption peaks. Comparison of the spectral characteristics of the isolated pigments with OF indicate that almost all of the measured chloroplast pigments of Chrysochron~ulina polylepis transfer light-energy to photosystem 11. This is in agreement with Haxo's survey of photosynthetic effectiveness in the similar pigmented Emilianla huxleyi, based on spectral absorption, fluorescence excitation and 02-action spectrum (Haxo 1985).

6 246 Mar. Ecol. Prog. Ser. 83: , 1992 Table 3. Chrysochromulina polylepis and Skeletonema costatum. Photosynthetic parameters in 'blue-green' incubator light and cell chemistry (data for S. costatum: M. Gilstad & G. Johnsen unpubl.). Photosynthetic parameters: P:, realized maximum uptake; P:, theoretical rnax~mum uptake; a', photosynthetic efficiency; BB, photoinhibition ~ndex; Ik (=P:/&'), photoadaptation index; I,,, optimum irradiance; Ib, photo~nhibition irradiance. "&: spectrally corrected absorbed quanta; 'F. absorbed quanta transported to photosystem I1 (see Eq. 1). Superscript C denotes carbon-normalized coefficients Parameter C polylepis S. costatum Unit I, Ib - Oa,, "F Chl a:c N:C CP. =pm mg C (mg chl a)-' h-' mg C (rng chl a)-' h-' mg C (mg chl a)-' h-' (pm01 m-' S-')-' mg C (mg chl a)-' h-' (prnol m-2 S-')-' pm01 m-2 S-' pm01 m-' S-' pm01 m-2 S-' m2 (rng chl a)-' w:w at..at. m2 (mg C)-' h-' h-' h-' (pm01 m-' S-')-' "a, and "F differ relatively little whether Chrysochromulina polylepis or Skeletonema costa tum are concerned - presumably as a result of the small content of photoprotective pigments, namely diadino- and diatoxanthin, in shade-adapted cells (Vernet et al. 1989, Sakshaug et al. 1991). While "F of C. polylepis exhibits a maxlmum at 472 nm, "F of S. costatum exhibits a maximum at 440 nm. Thus C. polylepis is somewhat more specialized than S. costatum in absorption of blue-green light (Figs. 2B, C & 3A). Light harvesting Based on chl a-normalization, Chrysochromulina polylepis absorbs blue-green incubator light 1.7 times more efficiently than Skeletonema costatum, whether in terms of "For "ZC(Table 3). This is related to the large amounts of 19'-hexanoyloxyfucoxanthin, chl c2 and chl c3 relative to chl a (Fig. 3). Since the fucoxanthin: chl a ratio in both species (fucoxanthin + 19'-hexanoyloxyfucoxanthin in C. polylepis) is about 0.5, the large difference in chl a-normalized spectra between species must be due to particularly efficient light-harvesting pigment-protein bonds in C. polylepis. The shoulder at 592 nm in both "a, and "F obtained from C. polylepis indicates that chl c2.+ are efficient light-harvesting pigments (Figs. 2B, C & 3). C. polylepisis more efficient than S. costatum not only because these pigments transfer light-energy to photosystem 11 extremely efficiently, but also because it has 1.36 times more of total chl c per unit chl a (Fig. 3A, Table 2). C. polylepis, however, has an 2 value in blue-green incubation Light which is only 1.2 times higher than in S. costatum. The 1.4 times higher value of Chrysochromulina polylepis relative to Skeletonema costaturn for "ac at 676 nm, in which absorption by accessory pigments is negligible, indicates that the 'packaging' effect, i.e. self-shading in and between chloroplasts (Kirk 1983), is important in S. costatum. This is presumably related to the twice as high chl a: C ratio in S. costatum relative to C. polylepis (Table 3). The 2.2 times higher value for "a, (440 nm) in C. polylepis compared to S. costatum reflects that accessory pigments are more important in live cells of C. polylepis than in live cells of S. costatum (Flg. 3A). Light harvesting vs spectral irradiance To demonstrate the effect of spectral distribution on algal photosynthesis in low light, we calculated "F, the absorbed quanta transported to photosystem I1 (Eq. l), of Chrysochromulina polylepis and Skeletonema costatum in green and blue waters, assuming infinitesimal chl a concentrations (Table 4). It turns out that both species, in spite of being coastal strains, absorb light more efficiently in blue than in green water (Fig. 2B, C). Per unit chl a, C. polylepis absorbs quanta transported to photosystem I1 ("F) 1.96, 1.86 and 1.71 times more efficiently than S. costatum in green coastal water at 5, 20 and 50 m depth, respectively, and 2.07 to 2.13 times more efficiently than S. costatum In blue water (Table 4, Fig. 2B). 2 corresponds to the product of G,,,,

7 Johnsen et al.: Photobiology of Chrysochromulina polylepis Table 4. Chrysochrornulina polylepis (Cp) and Skeletonerna has a low P: and Ik well below the growth irradiance costaturn (Sc). Spectrally corrected transport of absorbed_ used in our experiments, in addition to an extremely quanta to photosyste~ I1 (Eq. l), based on data from Fig. 2; "F low value for carbon-normalized maximum carbon [m2 (mg chl a)-'], 'F [m2 (mg C)-' x 103] in green coastal waters (G) and blue oceanic waters (B; from Smith & Baker uptake (=P,; Table 3), the opposite is true for shade- 1981) at 5, 20 and 50 m depth, assuming infinitesimal chl a adapted S. costatum. This, and the high susceptibility concentration of C. polylepis to photoinhibition, may imply that shade-adapted C. polylepis, in contrast to Skeletonema - Water, "F "F "F(Cp) 'F 'F "F(Cp) costatum, cannot tolerate short periods of strong light depth (CP) (Sc) OF (Sc) (CP) (Sc) CF(Sc) well. It may, however, compensate for this by phototaxis. Phototactic compensation is only possible in well- G, 5 m stratified water layers, such as during the bloom of C. G, 20 m polylepis in KattegaUSkagerrak in Conse- G, 50 m quently, the 2 species, when shade-adapted, may differ B, 5 m primarily in their response to strong light. B, 20 m Although not measured here, the growth rate of B, 50 m Chrysochrornulina polylepis should be low. Our set of photosynthetic coefficients implies a gross growth rate (net rate, + respiration rate, r) of 0.46 d-' at 75 pm01 which can be considered as constant in shade-adap- m-' S-' and 16 h day length, in contrast to 1.26 d-' for tated cells (Langdon 1988), and the spectrally corrected Skeletonema costatum according to studies by Saktransport of light-energy to photosystem I1 ("F). As a shaug et al. (1989). Correspondingly, the net growth consequence, 2 should vary in a linear proportion with rate (p) of C, polylepis should be half or less than the "F(Table 4; Johnsen & Hegseth 1991). Per unit carbon, value of about 0.8 d-' for S. costatum, unless the however, absorption of light ('F and 'a,) by C. poly- species differ profoundly in terms of the respiration rate lepis is actually a little smaller than in S. costatum in (Sakshaug & Andresen 1986). green water while being equal in blue water (Table 4, Fig. 2C). Because growth 1s related to carbon-normalized parameters, a high value of the chlorophyll- CONCLUSIONS normalized factor "F can be deceptive in the context of growth rate; i.e. the high "F of C. polylepis is offset by The high efficiency of Chrysochromulina polylepis the high chl a: C ratio in S. costatum. One should bear with respect to light harvesting in blue-green to green in mind, however, that to compare with S. costatum is waters (Table 4) may have played a role in its success to compare with the most successful diatom species in during the initial phase of the bloom in the Kattegat/ nearshore Norwegian coastal waters and fjords (see Skagerrak in summer Moreover, low surface Sakshaug & Andresen 1986). In fact, one may conclude salinity, high surface temperature and the resulting that both species are efficient in absorption of blue as stratification of the upper layers, as well as unusually well as blue-green light (see Table 4). sunny weather, may presumably have ensured close to ideal conditions for this species (Aksnes et al. 1989, Skjoldal & Dundas 1991). Similar conditions have also Photosynthetic parameters favoured another Chrysochromulina bloom, i.e. of the toxic Chrysochromulina leadbeaten mixed with C. The photosynthetic parameters in Chrysochromulina encina, C. hirta and peridinin-containing dinoflagelpolylepis indicate relatively high susceptibility to lates in the surface layers in the Vestfjord/Tysfjord area photoinhibition compared to shade-adapted diatoms in northern Norway in May-June 1991 (Johnsen 1991). (Richardson et al. 1983). High susceptibility to strong Our calculations indicate that Skeletonema costatum light, however, was not observed in field samples would grow faster than Chrysochrornulina polylepis, (Nielsen et al. 1990). They observed assimilation num- particularly in the medium-to-low light near the pycnobers from 0.6 to 6.7 mg C (mg chl a)-' h-' for C. cline, considering the different carbon-specific photopolylepis obtained from the pycnocline and illuminated synthetic efficlencies (=a,) of the 2 species (Table 3). at an irradiance corresponding to 2.5 m depth. Such Because a majority of 'nuisance' species appear to differences in assimilation numbers are likely to be due grow slower than many diatoms in comparable light to difference in incubation technique, temperature, regimes (Brand & Guillard 1981), one might justifiably photoadaptation (Sakshaug et al. 1991), or different ask why S. costatum and other fast-growing species are isolates may be inherently different physiologically. absent during some phytoplankton blooms, rather than While shade-adapted Chrysochromulina polylepis why C. polylepis and related species are successes.

8 248 Mar. Ecol. Prog. Ser. 83: This question, however, cannot be answered in terms of light strategies alone. Initial stocks of competitive species, for instance, may have been too small or absent, and a low supply of silicate may have controlled the diatom stock, at least late in the bloom of C. polylepis (Aksnes et al. 1989, Skjoldal & Dundas 1991). Moreover, whereas diatoms such as Skeletonema are bound to sink and to be grazed to a considerable extent, C. polylepis may stay near the pycnocline due to its motility while not being grazed appreciably due to its toxicity (Nielsen et al. 1990). Acknowledgements. This work was supported by The Royal Norwegian Council for Scienhfic and Industrial Research (G.J.) and The Norwegian Research Council for Science and the Humanities (M.V.). Thanks are due to M. V Nielsen and L. Granskog for growing C polylepis, M. Gilstad for use of unpublished data, and Kjersti Andresen for drawing the figures. We also thank Prof. E. Paasche and S Myklestad for supplying iso!;:es of C. polylepis and S. costatum, respectively. Contribution 251, Trondhjem Biological Station. LITERATURE CITED Aksnes, D. I., Aure, J.. Furnes, G. K., Skjoldal. H. R., Setre. R. (1989). Analyse av Chrysochromulina polylepis - oppblomstringen i Skagerrak, mai Milj~rbetingelser og mulige irsaker In: Draget, H. B. D., Holthe, T (eds.) Oppblomstring av Chrysochromulina polylepis 1988, Report 12. Directorate for Nature Management Norway, p (in Norwegian) Bjerkeng, B.. Vernet, M., Nielsen, M.V., Liaaen-Jensen, S. (1990). Carotenoids of Chrysochromulina polylepis (Prymnesiophyc~ae). Biochem. Syst. Ecol Brand, L. E, Guillard, R. R. L. (1981). The effects of continuous light and light intensity on the reproduction rates of twenty-two species of marine phytoplankton. J. exp. mar. Biol. Ecol. 50: Dahl, E., Lindahl, O., Paasche, E., Throndsen, J. (1989). The Chrysochromulina polylepis bloom in Scandmavian waters during spring In: Cosper. E. M,, Bricell. V. M,, Carpenter, E. J. (eds.) Novel phytoplankton blooms. Coastal and Estuarine Studies 35. Springer Verlag, Heidelberg, p Davies, B H (1976). Carotenolds. In: Goodwin, T W (ed.) Chemistry and biochemist? of plant pigments. Academic Press, New York, p Fawley, M. W. (1989). Detection of chlorophylls c,, c2 and cl in pigment extracts of Prymnesium parvum (Prymnesiophycede). J Phycol. 25: Gieskes, W. W. C., Kraay, G W (1986) Anal.ysis of phytoplankton pigments by HPLC before, during and aftcr mass occurrence of the mlcroflagellate Conmbellus aureus during the spring bloom in the open North Sea in mar. Biol. 92: Graneli, E., Carlsson, P., Olsson, P,, Sundstrom, B., Granell, W., Llndahl, 0. (1989) From anoxia to fish poisoning: the last ten years of phytoplankton blooms in Swedlsh marine waters. In: Cosper, E. hl., Bricelj, V M,, Carpenter, E. J. (eds.) Novel phytoplankton blooms. Coastal and Estuarine Studies 35. Springer Verlag, Heidelberg, p Gulllard, R. R L., Ryther. J. H. (1962) Studies of rnar~ne plankton diatoms. 1. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8: Hallegraeff, G. M. (1981). Seasonal study of phytoplankton pigments and species at a coastal station off Sydney: importance of diatoms and the nanoplankton. Mar Biol. 61: Haxo, F. T (1985). Photosynthetic action spectrum of the coccolithphorid, Emiliania huxleyi (Haptophyceae): 19'hexanoyloxyfucoxanthin as antenna pigment. J Phycol Jeffrey, S. W (1968). Quantitative thin-layer chromatography of chlorophylls and carotenoids from marine algae. Biochim. Biophys. Acta. 162: Jeffrey, S. W., ~ikphre~, G. F. (1975). New spectrophotometnc equations for determining chlorophylls a, b, c, and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz. 167: Jeffrey, S. W., Wright, S. W (1987). A new spectrally distinct component in preparations of chlorophyll cfrom the microalga Emiliania huxleyl (Prymnesiophyceae) Biochim. Biophys. Acta 894: Jerlov, N. G. (1976). Marine opt~cs. 2nd edn. Elsevier,.4msterdam Johnsen, G. (1991). Identification of Chrysochromulina leadbeateri by means of remote sensing. Fisken og Havet (in Norwegian with English summary) Johnsen, G., Hegseth, E. N. (1991). Photoadaptat~on of sea-ice microalgae in the Barents Sea. Polar Biol Kaartvedt, S., Johnsen, T. M., Aksnes, D. L., Lie, U., Svendsen, H. (1990). Giftalgen Prymnesium parvum i Ryfylkefjor- dene, juli-august IMB Rapport 2: University of Bergen, Norway, p 1-68 (in Norwegian) Kirk, J. T 0. (1983). Light and photosynthesis in aquatic ecosystems. Cambridge University Press, Cambridge Kopf, U,, Heinze, J. (1984). 2.7-bis(diethy1amino)phenazoxonium chloride as a quantum counter for emission measurements between 240 and 700 nm. Analyt. Chem. 56: Langdon. C. (1988). On the causes of interspecific differences in the growth-irradiance relationship for phytoplankton. 11. A general review. J. Plankton Res. 10: Lewis, M. R., Smith, J. C. (1983) A small volume, shortincubation time method for measurement of photosynthesis as a function of incident irradiance. Mar. Ecol. Prog. Ser. 13: Lindahl, O., Dahl, E. (1989). On the development of the Chrysochromulina polylepis bloom in the Skagerrak in May- Junr In Graneli, E., Sundstrom, B., Edler, L., Anderson, D, hf. (eds.) Toxic marine phytoplankton. Elsevi.er, Amsterdam, p Manton, J., Parke, M. (1962). Preliminary observations on scales and their mode of origin in Chrysochromulina polylepis sp. nov J mar. biol. Ass U.K. 42: Mantoura, R. F. C., Lleurellyn, C A. (1983). The rapid determination of algal chlorophyll and carotrnoid plgments and their breakdown products in natural waters by reverse- phase high-pressure liquid chromatography. Analyt. Chim. Acta Mitchell, B. G., Kiffer, D. A. (1988). Chlorophyll a specificabsorption and fluorescence excitation spectra for lightlimited phytoplankton. Deep Sea Res Morel, A., Lazzara, L., Gostan, J. (1987). Growth rate and quantum yleld time response for a diatom to changing irradiance (energy and color). Lmnol. Oceanogr. 32: Nelson, J. R., Wakeham, S G. (1989). A phytol-substituted

9 Johnsen et al.: Photobiology of Chrysochromulinil polylepjs 249 chlorophyll c from Emiliana huxleyi (Prymnesiophyceae) J. Phycol. 25: Neori, A., Vernet, %I., Holm-Hansen, O., Haxo, F. T (1988). Comparison of chlorophyll far-red fluorescence excitation spectra with photosynthetic oxygen action spectra for photosystem 11 in algae. Mar Ecol. Prog Ser 44 2 ~ 302 i Nielsen, T G., Kiarboe, T., Bjarnst-n, P. K. (1990) Effects of a Chrysochron~ulina polylepis subsurface bloom on the plankton con~munity. Mar Ecol. Proq Srr Pennington, F., Cuillard, R. R. L., Liaaen-Jensen, S. (1988). Carotenoid distribution patterns in Bacillariophyceae (Diatoms). Biochem. Syst. Ecol Platt, T., Gallegos, C. L., Harrison, W. G. (1980) Photo-inhibition of photosynthesis in natural assemblages of marine phytoplankton. J. mar Res. 38: Rchardson, K., Beardall, J., Raven, J. A. (1983). Adaptation of unicellular algae to irradiance: an analysis of strategies. New Phytol. 93: Riley, J. P., Wilson, T. R. S. (1967). The pigments of some marine phytoplankton species. J. mar. biol. Ass. U.K. 47: Sakshaug, E., Andresen, K. (1986). Effect of light regime upon growth rate and chemical composition of a clone of Skeletonema costatum from the Trondheimsfjord. Norway. J. Plankton Res. 8: Sakshaug, E.. Andresen, K., Kiefer, D. A. (1989). A steady state description of growth and light absorption in the marine diatom Skeletonen~a costaturn. Limnol. Oceanogr Sakshaug, E, Johnsen. G., Andresen, K., Vernet, M. (1991). Modeling of light-dependent algal photosvnthesis and growth experiments with the Barents Sea diatoms Thalassloslra nordenskioeldii and Chaetoceros furc~ll~~tus. Deep Sea Res Skloldal, H R, Dundas, I. (eds). (1991). The Chrysochron~uhna polylepis bloom in the Skagerrak and the Kattegat in May-June 1988: environmental conditions, possible causes, and effects. Coop. Res. Rep. Int. Counc. Explor. Sea (ICES) Workshop. No pp. Smith, R. C., Baker, K. S. (1981). Optical properties of the clearest natural waters ( nm). Appl. Optics 20: Vernet, M., Neon, A., Haxo, F. T (1989). Spectral properties and photosynthetic action in red-tide populations of Prorocentrum micans and Gonyaulax polyedra. Mar Biol. 103: Wright, S. W.. Jeffrey, S. W. (1987). Fucoxanthin pigment markers of marine phytoplankton analysed by HPLC and HPTLC. Mar. Ecol. Prog. Ser 38: Wright. S. W., Shearer. J. D. (1984). Rapid extraction and highperformance liquid chromatography of chlorophylls and carotenoids from marine phytoplankton. J. Chromatogr 294: This article was presented by H. R. Skjoldal, Bergen, Norway manuscript first received: May 14, 1991 Revised version accepted: April 21, 1992